MVA originates from the dermal vaccinia strain Ankara (Chorioallantois vaccinia Ankara (CVA) virus) that was maintained in the Vaccination Institute, Ankara, Turkey for many years and used as the basis for vaccination of humans. However, due to the often severe post-vaccinal complications associated with vaccinia viruses (VACV), there were several attempts to generate a more attenuated, safer smallpox vaccine.
During the period of 1960 to 1974, Prof. Anton Mayr succeeded in attenuating CVA by over 570 continuous passages in CEF cells (Mayr et al., 1975, Passage History: Abstammung, Eigenschaften and Verwendung des attenuierten Vaccinia-Stammes MVA. Infection 3: 6-14). As part of the early development of MVA as a pre-smallpox vaccine, there were clinical trials using MVA-517 (corresponding to the 517th passage) in combination with Lister Elstree (Stickl, 1974, Smallpox vaccination and its consequences: first experiences with the highly attenuated smallpox vaccine “MVA”. Prev. Med. 3(1): 97-101; Stickl and Hochstein-Mintzel, 1971, Intracutaneous smallpox vaccination with a weak pathogenic vaccinia virus (“MVA virus”). Munch Med Wochenschr. 113: 1149-1153) in subjects at risk for adverse reactions from vaccinia. In 1976, MVA derived from MVA-571 seed stock (corresponding to the 571st passage) was registered in Germany as the primer vaccine in a two-stage parenteral smallpox vaccination program. Subsequently, MVA-572 was used in approximately 120,000 Caucasian individuals, the majority children between 1 and 3 years of age, with no reported severe side effects, even though many of the subjects were among the population with high risk of complications associated with conventional vaccinia virus (Mayr et al., 1978, The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behaviour in organisms with a debilitated defence mechanism (author's transl). Zentralbl. Bacteriol. (B) 167: 375-390). MVA-572 was deposited at the European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, as ECACC V9401 2707.
Being that many passages were used to attenuate MVA, there are a number of different strains or isolates, depending on the passage number in CEF cells. All MVA strains originate from Dr. Mayr and most are derived from MVA-572 that was used in Germany during the smallpox eradication program, or MVA-575 that was extensively used as a veterinary vaccine. MVA-575 was deposited on Dec. 7, 2000, at the European Collection of Animal Cell Cultures (ECACC) with the deposition number V001 20707.
By serial propagation (more than 570 passages) of the CVA on primary chicken embryo fibroblasts, the attenuated CVA-virus MVA (modified vaccinia virus Ankara) was obtained. MVA was further passaged by Bavarian Nordic and is designated MVA-BN. MVA, as well as MVA-BN, lacks approximately 13% (26.5 kb from six major and multiple minor deletion sites) of the genome compared with ancestral CVA virus. The deletions affect a number of virulence and host range genes, as well as a large fragment of the gene coding for A-type inclusion protein (ATI) and a gene coding for a structural protein directing mature virus particles into A-type inclusion bodies. A sample of MVA-BN was deposited on Aug. 30, 2000, at the European Collection of Cell Cultures (ECACC) under number V00083008.
MVA-BN can attach to and enter human cells where virally-encoded genes are expressed very efficiently. However, assembly and release of progeny virus does not occur. Preparations of MVA-BN and derivatives have been administered to many types of animals, and to more than 2000 human subjects, including immunodeficient individuals. All vaccinations have proven to be generally safe and well tolerated.
The perception from many different publications is that all MVA strains are the same and represent a highly attenuated, safe, live viral vector. However, preclinical tests have revealed that MVA-BN demonstrates superior attenuation and efficacy compared to other MVA strains (WO 02/42480). The MVA variant strains MVA-BN as, e.g., deposited at ECACC under number V00083008, have the capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF), but no capability of reproductive replication in human cells in which MVA 575 or MVA 572 can reproductively replicate. For example, MVA-BN has no capability of reproductive replication in the human keratinocyte cell line HaCaT, the human embryo kidney cell line 293, the human bone osteosarcoma cell line 143B, and the human cervix adenocarcinoma cell line HeLa. Further, MVA-BN strains fail to replicate in a mouse model that is incapable of producing mature B and T cells, and as such is severely immune compromised and highly susceptible to a replicating virus. An additional or alternative property of MVA-BN strains is the ability to induce at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes.
The term “not capable of reproductive replication” is used in the present application as defined in WO 02/42480 and U.S. Pat. No. 6,761,893, which are hereby incorporated by reference. Thus, the term applies to a virus that has a virus amplification ratio at 4 days after infection of less than 1 using the assays described in U.S. Pat. No. 6,761,893, which assays are hereby incorporated by reference. The “amplification ratio” of a virus is the ratio of virus produced from an infected cell (Output) to the amount originally used to infect the cells in the first place (Input). A ratio of “1” between Output and Input defines an amplification status wherein the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells.
MVA-BN or its derivatives are, according to one embodiment, characterized by inducing at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes. A vaccinia virus is regarded as inducing at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes if the CTL response as measured in one of the “assay 1” and “assay 2” as disclosed in WO 02/42480, preferably in both assays, is at least substantially the same in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes. More preferably, the CTL response after vaccinia virus prime/vaccinia virus boost administration is higher in at least one of the assays, when compared to DNA-prime/vaccinia virus boost regimes. Most preferably, the CTL response is higher in both assays.
WO 02/42480 discloses how vaccinia viruses are obtained having the properties of MVA-BN. The highly attenuated MVA-BN virus can be derived, e.g., by the further passage of a modified vaccinia virus Ankara (MVA), such as MVA-572 or MVA-575.
In summary, MVA-BN has been shown to have the highest attenuation profile compared to other MVA strains and is safe even in severely immunocompromised animals.
Although MVA exhibits strongly attenuated replication in mammalian cells, its genes are efficiently transcribed, with the block in viral replication being at the level of virus assembly and egress. (Sutter and Moss, 1992, Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. U.S.A. 89: 10847-10851; Carroll and Moss, 1997, Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238: 198-211.) Despite its high attenuation and reduced virulence, in preclinical studies MVA-BN has been shown to elicit both humoral and cellular immune responses to VACV and to the products of heterologous genes cloned into the MVA genome (Harrer et al., 2005, Therapeutic Vaccination of HIV-1-infected patients on HAART with recombinant HIV-1 nef-expressing MVA: safety, immunogenicity and influence on viral load during treatment interruption. Antiviral Therapy 10: 285-300; Cosma et al., 2003, Therapeutic vaccination with MVA-HIV-1 nef elicits Nef-specific T-helper cell responses in chronically HIV-1 infected individuals. Vaccine 22(1): 21-29; Di Nicola et al., 2003, Clinical protocol. Immunization of patients with malignant melanoma with autologous CD34(+) cell-derived dendritic cells transduced ex vivo with a recombinant replication-deficient vaccinia vector encoding the human tyrosinase gene: a phase I trial. Hum Gene Ther. 14(14): 1347-1 360; Di Nicola et al., 2004, Boosting T cell-mediated immunity to tyrosinase by vaccinia virus-transduced, CD34(+)-derived dendritic cell vaccination: a phase I trial in metastatic melanoma. Clin Cancer Res. 10(16): 5381-5390.)
MVA-BN and recombinant MVA-BN-based vaccines can be generated, passaged, produced and manufactured in CEF cells cultured in serum-free medium. Many recombinant MVA-BN variants have been characterized for preclinical and clinical development. No differences in terms of the attenuation (lack of replication in human cell lines) or safety (preclinical toxicity or clinical studies) have been observed between MVA-BN, the viral vector backbone, and the various recombinant MVA-based vaccines.
Induction of a strong humoral and cellular immune response against a foreign gene product expressed by a VACV vector is hampered by the fact that the foreign gene product has to compete with all of the more than 150 antigens of the VACV vector for recognition and induction of specific antibodies and T cells. The specific problem is the immunodominance of vector CD8 T cell epitopes which prevents induction of a strong CD8 T cell response against the foreign gene product. (Smith et al., Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara. J. Immunol. 175:8431-8437, 2005.) This applies to replicating VACV vectors such as Dryvax, as well as for non-replicating vectors like NYVAC and MVA.
For expression of a recombinant antigen (“neoantigen”) by VACV, only poxvirus-specific promoters, but not common eukaryotic promoters, can be used. The reason for this is the specific biology of poxviruses which replicate in the cytoplasm and bring their own, cell-autonomous transcriptional machinery with them that does not recognize typical eukaryotic promoters.
The viral replication cycle is divided into two major phases, an early phase comprising the first two hours after infection before DNA replication, and a late phase starting at the onset of viral DNA replication at 2-4 hours after infection.
The late phase spans the rest of the viral replication cycle from ˜2-20 h after infection until progeny virus is released from the infected cell. There are a number of poxviral promoter types which are distinguished and named by the time periods within the viral replication cycle in which they are active, for example, early and late promoters. (See, e.g., Davison and Moss, J. Mol. Biol. 210:771-784, 1989; Davison and Moss, J. Mol. Biol. 210:749-769, 1989; and Hirschmann et al., Journal of Virology 64:6063-6069, 1990, all of which are hereby incorporated by reference.)
Whereas early promoters can also be active late in infection, activity of late promoters is confined to the late phase. A third class of promoters, named intermediate promoters, is active at the transition of early to late phase and is dependent on viral DNA replication. The latter also applies to late promoters, however, transcription from intermediate promoters starts earlier than from typical late promoters and requires a different set of transcription factors.
It became increasingly clear over recent years that the choice of the temporal class of poxviral promoter for neoantigen expression has profound effects on the strength and quality of the neoantigen-specific immune response. It was shown that T cell responses against neoantigens expressed under the control of a late promoter are weaker than those obtained with the same antigen under an early promoter. (Bronte et al., Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine. Proc. Natl. Acad. Sci. U.S. A 94:3183-3188, 1997. Coupar et al., Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response. Eur. J. Immunol. 16:1479-1487, 1986.)
Even more strikingly, it was recently shown that in repeated autologous immunizations with VACV as well as with the replication-defective VACV vector MVA, CD8 T cell responses against antigens under an exclusively late promoter can fail completely. This failure resulted in an almost undetectable antigen-specific CD8 T cell response after the second immunization. (Kastenmuller et al., Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination. J. Exp. Med. 204:2187-2198, 2007.)
Thus, early expression of neoantigens by VACV vectors appears to be crucial for efficient neoantigen-specific CD8 T cell responses. It has also been shown that an early-expressed VACV vector antigen not only competes with late expressed antigens but also with other early antigens for immunodominance in the CD8 T cell response. (Kastenmuller et al., 2007.) The specific properties of the early portion of the poxviral promoter might thus be very important for induction of a neoantigen-specific T cell response. Moreover, it is a commonly held view and a general rule that higher amounts of antigen are beneficial for induction of stronger antigen-specific immune responses (for the poxvirus field, see for example Wyatt et al., Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26:486-493, 2008).
A promoter combining 4 early promoter elements and a late promoter element from the ATI gene has been described previously (Funahashi et al., Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses. J. Virol. 65:5584-5588, 1991; Wyatt et al., Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26:486-493, 2008), and has been shown to direct increased early expression of antigen. However, T cell responses induced by an antigen driven by such a promoter have only been analyzed after a single immunization and were not apparently different from those obtained with the classical Pr7.5K promoter in this setting. (Funahashi et al., Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses. J. Virol. 65:5584-5588, 1991.)
Jin et al. Arch. Virol. 138:315-330, 1994, reported the construction of recombinant VACV promoters consisting of a VACV ATI promoter combined with tandem repeats (2 to 38 copies) of a mutated Pr7.5 promoter operably linked to the CAT gene. Up to 10 repetitions of the mutated Pr7.5 promoter were effective in increasing early gene expression. Further repetition appeared to be inhibitory. With all constructs, the amount of CAT protein produced in the presence of cytosine arabinoside (AraC) (i.e. when the viral replication cycle was arrested in the early phase) was less than one-tenth of the amount produced in the absence of AraC (Jin et al. Arch. Virol. 138:315-330, 1994).
Recently, it was shown that repeated immunizations of mice with recombinant MVA expressing OVA under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element led to superior acute and memory CD8 T-cell responses compared to those to Pr7.5- and PrS-driven OVA. Baur et al., Journal of Virology, Vol. 84 (17): 8743-8752 (2010). Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. Id.
Assarsson et al., P.N.A.S. 105: 2140-45, 2008, simultaneously measured the expression levels of 223 annotated vaccinia virus genes during infection and determined their kinetics using a genome tiling array approach. They found that many genes in the WR strain of Vaccinia virus had high transcription rates. Assarsson et al. provided some examples of highly expressed genes: immediate-early, VACWR-059 (double-stranded RNA-binding protein) and VACWR-184 (unknown); early, VACWR-018 (unknown); early/late, VACWR-131 (core protein); and late, VACWR-169 (unknown). Assarsson et al. indicated that, because of their exceptionally high expression levels, these genes might be of special interest for future investigations, but did not identify the promoters initiating transcription of these genes.
Yang et al., P.N.A.S. 107:11513-11518, 2010, used deep RNA sequencing to analyze vaccinia virus (VACV) transcriptomes at progressive times following infection. Before viral DNA replication, transcripts from 118 VACV ORFs were detected; after replication, transcripts from 93 additional ORFs were characterized. The high resolution permitted determination of the precise boundaries of many mRNAs including read-through transcripts and location of mRNA start sites and adjacent promoters.
Orubu et al, PLoS ONE 7(6):e40167, 2012, showed that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5.
Strong T cell and antibody responses against antigens encoded by recombinant poxviruses can improve vaccine efficacy. Consequently, a need in the art exists for compositions and methods capable of achieving strong T cell and antibody responses against antigens encoded by recombinant poxviruses, such as MVA. The invention fulfills this need.